Virtually all organisms have nuclease enzymes that degrade rapidly foreign DNA as an important in vivo defense mechanism. The use, therefore, of normal oligonucleotides as diagnostic or therapeutic agents in the presence of most bodily fluids or tissue samples is generally precluded. It has been shown, however, that phosphoromonothioate or phosphorodithioate modifications of the DNA backbone in oligonucleotides can impart both nuclease resistance and enhance the affinity for target molecules, such as for example the transcriptional activating protein NF-κB.
Recent world events have heightened the awareness of possible bioterrorist threats. Hemorrhagic fever viruses (category A bioweapon agents) have reportedly been weaponized by the former Soviet Union and the United States (Borio et al., 2002; Hawley & Eitzen, 2001). Despite the awareness of the potential of Viral Hemorrhagic Fever viruses (Lassa, Junin), Encephalitic viruses (West Nile, VEE) and other agents both as bioweapons and as emerging viral diseases, few therapeutic options are available to those infected. Apart from supportive therapy, the only drug for treating Arenavirus infections is Ribavirin and it is only partially effective (McCormick et al, 1986a; Shulman, 1984; Enria et al., 1987) while there are no efficacious drugs to treat victims of West Nile infections (Peterson and Marfin, 2002). There is an urgent need to expand the current therapeutic armamentarium, which is hindered, at least in part, by a lack of in-depth knowledge concerning the mechanisms of Arenaviral pathogenesis (Peters & Zaki, 2002).
Arenavirus pathogenesis stems from host immune response dysregulation and endothelial dysfunction (Peters & Zaki, 2002; Ignatyev et al., 2000; McCormick & Fisher-Hoch, 2002; Walker et al., 1982; McCormick et al., 1986b; Marta et al., 1999). West Nile pathogenesis is associated with the inability of host immune response to limit virus replication to levels below that required for viral invasion of the CNS (Solomon and Vaughn, 2002).
Lassa fever, a human arenavirus hemorrhagic fever virus endemic in West Africa, affects up to 300,000 people annually and is responsible for up to 3000 deaths (McCormick, et al., 1987). Lassa Fever virus is difficult to study due to its hazardous nature (a BSL4 agent). Junin Virus is the causative agent of Argentine hemorrhagic fever (AHF). The annual incidence varies between 100-4000 cases/yr. AHF has a case fatality rate of 15-30% and is also a BSL4 agent. A well-established animal model that resembles Lassa Fever, using the non-pathogenic New World Arenavirus, Pichinde virus (Jahrling et al., 1981) has been used to study this class of pathogens. Serial passage of Pichinde virus in guinea pigs was used to develop a virulent variant that produces a disease in guinea pigs that mimics human Lassa Fever in many important respects including: viremia correlates with disease outcome (Johnson et al., 1987; Aronson et al., 1994), a relative paucity of pathologic findings in lethally infected animals (Walker, et al., 1982; Connolly, et al., 1993), terminal vascular leak syndrome (Katz & Starr, 1990) and distribution of viral antigens within the host (Connolly et al., 1993; Shieh et al., 1997; Aronson, unpublished data). Macrophage responses to the attenuated Pichinde virus, P2, with the virulent Pichinde variant, P18 as well as reassortants of the two variants (Zhang et al., 1999; Zhang et al., 2001; Fennewald et al., 2002) may be used to compare and modify the immune response to viral infection.
West Nile virus (Category B virus) is a mosquito-borne flavivirus that is a neuropathogen in humans, equines and avians (Solomon and Vaughn, 2002; Petersen and Marfin, 2002). Humans become infected by the bite of an infected mosquito. The viruses are then thought to replicate in the skin before being transported to the local lymph nodes. West Nile may then spread via the blood to other organs including the liver, spleen, heart and kidney and eventually the brain. West Nile virus may spread to the CNS via either hematogenous spread or via the olfactory mucosa where there is no blood-brain barrier. West Nile is an emerging pathogen in the US, spreading across the country since it was first identified in New York in 1999. As of Oct. 3, 2002, the CDC has reported 2530 cases of West Nile virus infection with 125 deaths in 32 states. West Nile is also responsible for major outbreaks in other countries including Tunisia, Romania, Algeria, Russia and Israel among others. Case fatality rates range from 4-29%. Age is a risk factor in the development of severe West Nile disease with many patients exhibiting substantial morbidity. Presently, treatment for West Nile is limited to supportive intervention. There is no evidence that either interferon or Ribavirin treatment is efficacious (Petersen and Marfin, 2002).
Arenavirus Hemorrhagic Fevers, such as Lassa fever, Junin, Argentine hemorrhagic fever, Bolivian hemorrhagic fever and Venezuelan hemorrhagic fever, have several features in common with sepsis and the systemic inflammatory response syndrome, including fulminant clinical course, fever, shock, capillary leak syndrome, decreased myocardial contractility, abnormalities of coagulation and platelet function, and elevated serum levels of TNFα (Aronson et al., 1994; Cummins, 1990). Arenaviruses are non-cytopathic viruses with a tropism for macrophages and other reticuloendothelial cells (Cummins, 1990; Peters et al., 1987); the pathogenesis of these diseases is believed to involve excessive production of pro-inflammatory cytokines (Aronson et al., 1995; Peters et al., 1987). Unpublished data (Bausch et al., CDC) show cytokines to be massively activated in human Lassa fever, and also confirm that Lassa virus can directly induce cytokine secretion by infecting human macrophages in vitro (Mahanty et al., CDC, unpublished). Alternatively, there is evidence that a swift elaboration of pro-inflammatory cytokines and early engagement of the (innate) immune response may help protect of the infected host from lethal disease in various hemorrhagic fever syndromes (Peters et al., 1987).
Endotoxic shock results from an innate, anaphylactic response to bacterial lipopolysaccharide (LPS). The NF-κB transcription factor, in conjunction with other cellular transcription factors, plays a critical role in gene activation, especially in acute phase and inflammatory responses (Baeuerele, 1998; Barnes and Karin, 1997), and in particular endotoxic shock, a complex pathophysiological state which is considered to be an exaggerated or dysregulated systemic acute inflammatory response syndrome initiated by the binding of bacterial LPS complexed with lipopolysaccharide binding protein (LBP) to the CD14 receptor on macrophages. A series of intracellular signaling events, in which NF-κB activation figures importantly leads to enhanced transcription of proinflammatory mediators, including TNFα, IL-1 and inducible nitric oxide synthase, ultimately promoting vasodilatation, capillary leakiness, and myocardial suppression (Murphy et al., 1998). In well-established mouse endotoxemia models, rapid transient increases in NF-κB DNA-binding activity can be detected in the nuclei of macrophages and other cell types (Boher, et al., 1997); similar observations have been made in human sepsis (Velasco et al., 1997).
The AP-1 transcription factor family include the dimeric basic region leucine zipper proteins that belong to the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2) Maf (c-Maf, MafB, MafA, MafG/F/K, Nrl) and ATF/CREB (CREB, CREBP-2, ATF1, ATF2, LRF1/ATF3, ATF4, ATFa, ATF6, B-ATF, JDP1, JDP2) subfamilies which recognize either 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (5′-TGAG/CTCA-3′) or cAMP response elements (CRE, 5′-TGACGTCA-3′) (Chinenov and Kerppola, 2001; Shaulian and Karin, 2002). These transcription factor binding sites are elements in the promoters and enhancers of numerous mammalian genes including IL-2, IL-3, IL-4, IL-5, IFNβ, TNFα and GM-CSF (Chineov and Kerppola, 2001). The c-Jun protein is the most potent transcription factor. The c-Fos proteins, which cannot homodimerize can form heterodimers with c-Jun and thereby enhance their DNA binding activities. The c-Fos, and FosB proteins contain transactivation domains, however, Fra1, Fra2 and some splice variants of FosB do not. CREB and ATF1 can form homodimers and heterodimers but do not combine with other ATF proteins. ATF2, ATFa, CREBP-2, ATF3, ATF4 and ATF6 combine both with themselves and with specific Jun and/or Fos family members. C-Fos and Fra1 can heterodimerize with ATF4, but not with ATF2 and ATF3.
There are numerous other possible homodimers and heterodimers possible among this large group of BZIP proteins. Jun, Fos and ATF family members can also bind to DNA upon association with certain Maf, C/EBP and non-bZIP member factors like NF-κB, NFAT and Smad. This can direct AP-1 components to promoter sequences that only slightly resemble consensus AP-1 and ATF motifs. This variation in dimer partner and DNA binding site specificity is assumed to provide AP1 subunits with a high level of flexibility in gene regulation. The regulation of AP-1 family of transcription factor activity is complex but briefly regulation occurs through: 1) changes in jun and fos gene transcription and mRNA turnover, 2) Fos and Jun protein turnover, 3) post-translational modifications of both Fos, Jun other family proteins that modulate their activities, and 4) interactions with other transcription factors (Shaulian and Karin, 2001,2002). AP-1 activity is induced by growth factors, cytokines, neurotransmitters, polypeptide hormones, cell/matrix interactions, bacterial and viral infections and a variety of environmental stresses. These activators stimulate a series of signaling events that involve a variety of protein kinases including MAPKs, ERKs and JNKs. Members of the Fos and Jun protein families participate in the regulation of a variety of cellular processes including cell proliferation, differentiation, apoptosis, oncogenesis, inflammation, and immunity (Chinenov and Kerppola, 2001).